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Fig. 7 | Genome Biology

Fig. 7

From: A novel antiviral lncRNA, EDAL, shields a T309 O-GlcNAcylation site to promote EZH2 lysosomal degradation

Fig. 7

EDAL restricts viral replication by upregulation of Pcp4l1. a N2a cells were transfected with pcDNA3.1 or pcDNA-EDAL for 12 h and then infected with RABV at MOI 1 for 48 h. Total RNA was isolated and subjected to RNA-seq analysis (n = 2; 2 fold change (FC) and 0.01 p value). b N2a cells were transfected with pcDNA3.1 or pcDNA-EDAL for 48 h and then ChIP-seq analysis was performed. Volcano plot showed the peaks enriched in negative control (NC) cells and EDAL overexpression cells. The X axis was the log2 ratio of EDAL versus NC signals for each peak, and the Y axis was the significance of the differences (−log10 (p values)). c Six upregulated and loss of H3K27me3 mark genes were cloned into the mammalian expression vector pCAGGS and overexpressed in N2a cells. At 12 h post transfection, the cells were infected with RABV for 48 h at MOI 0.01, and virus titers in the supernatant were measured. d N2a cells were transfected with pCAGGS-Pcp4l1 (pC-Pcp4l1) at indicated dose for 12 h, and then infected with RABV at MOI 0.01. At 48 hpi, the virus load in the cell supernatant was measured. PCP4L1 expression level was analyzed by Western blotting. e N2a cells were transfected with siNC or three different sets of siPcp4l1 for 72 h and then PCP4L1 protein level was analyzed by Western blotting. f N2a cells were transfected with siNC or three different sets of siPcp4l1 for 24 h and then infected with RABV at MOI 0.01. At indicated hpi, virus load in cell culture was measured. g N2a cells were transfected with siNC or siPcp4l1–1 for 24 h and then transfected with pcDNA3.1 or pcDNA-EDAL for 12 h. Cells were then infected with RABV at MOI 0.01 and virus load in cell culture was measured at indicated hpi. h N2a cells were transfected with pCAGGS-Pcp4l1 (pC-Pcp4l1) for 12 h, and then infected with VSV at MOI 0.01. At indicated hpi, the virus load in the cell supernatant was measured. i N2a cells were transfected with pC-Pcp4l1 for 24 h, and then infected with SFV at MOI 0.01. At indicated hpi, the virus load in the cell supernatant was measured. j N2a cells were transfected with pC-Pcp4l1 for 24 h, and then infected with HSV-1 at MOI 0.01. At indicated hpi, the virus load in the cell supernatant was measured. k Sequencing profile of Pcp4l1 for ChIP-seq. The two tracks show H3K27me3 signals for pcDNA3.1 and pcDNA-EDAL samples after removing input background. The brown rectangle indicates the predicted promoter region of Pcp4l1. l N2a cells were transfected with pcDNA-EDAL or pcDNA3.1 for 48 h, and then ChIP-qPCR were performed with H3K27me3 antibody in the promoter region of Pcp4l1. m N2a cells were treated with 4 μM gsk126 or DMSO (mock) for 48 h and Pcp4l1 mRNA level was analyzed by qPCR. n Proposed model for EDAL-induced EZH2 lysosomal degradation, and the potential subsequent impact on EZH2-mediated epigenetic silencing of Pcp4l1. Statistical analysis of grouped comparisons was carried out by Student’s t test (**P < 0.01; ***P < 0.001). Bar graph represents means ± SD, n = 3. Western blot data are representative of at least two independent experiments

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